Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers’ rumen

Molecular cloning, purification, expression, and characterization of β-1, 4-endoglucanase gene (<i>Cel5A</i>) from <i>Eubacterium cellulosolvens</i> sp. isolated from Holstein steers’ rumen

Animal Bioscience, September 2017

28920412

Tansol Park, Seongwon Seo, Teaksoon Shin, Byung-Wook Cho, Seongkeun Cho, Byeongwoo Kim, Seyoung Lee, Jong K. Ha, Jakyeom Seo

This study was conducted to isolate the cellulolytic microorganism from a rumen of Holstein steer and characterize endoglucanase gene (Cel5A) from the isolated microorganism. To isolate anaerobic microbes having endoglucanase, the rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 99% similarity with Eubacterium cellulosolvens Ce2 (Accession number: AY178842.1). The Cel5A from isolated E. cellulosolvens sp. was cloned using the published genome sequence and expressed through the E. coli BL21. The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20 to 40°C but also, at the pH range of 3 to 9. The metal ions including K+, and Fe2+ significantly increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 µmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s-1) and 6.88 (mL/mg/s), respectively. Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of beneficial characteristics of this enzyme for possible industrial application.